Anti-acetaminophen antibodies and acetaminophen protein adducts

ABSTRACT

The present disclosure provides isolated antibodies that bind to acetaminophen-protein adducts that are useful in the detection and diagnosis of acetaminophen-induced toxicity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/086,923, filed Dec. 3, 2015, the disclosure of which is herebyincorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under R42 DK079387-03awarded by the NIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure provides isolated antibodies that bind toacetaminophen-protein adducts that are useful in the detection anddiagnosis of acetaminophen-induced toxicity.

BACKGROUND OF THE INVENTION

Acetaminophen (APAP) is the most common pharmaceutical productassociated with drug toxicity. In severe cases, APAP overdose may leadto acute liver failure (ALF) and death. Over 100,000 telephone callsconcerning APAP overdose are made to poison control centers in the U.S.annually. The FDA estimates that approximately 450 deaths are related toAPAP overdose annually. For patients that seek treatment within 24 hoursof an APAP overdose, and are able to provide accurate informationregarding the time and amount of APAP ingested, APAP overdose isrelatively straightforward to diagnose and treat. However, currentmethods of diagnosing APAP overdose, such as the Rumack nomogram, arenot very useful to diagnose patients after 24 hours of an APAP overdose,when information regarding time and dose of APAP ingested is notavailable, or patients that use alcohol, chronically ingestsupratherapeutic doses of APAP, or use sustained release APAPformulations. Other laboratory tests, such as serum alanineaminotransferase (ALT) and serum aspartate aminotransferase (AST),indicate the occurrence of liver damage, but neither bioindicator isspecific to APAP overdose.

Accordingly, a need exists in the art for a method of accuratelydiagnosing APAP toxicity, including occult APAP poisoning, even 24 hoursor longer after the overdose.

SUMMARY OF THE INVENTION

In an aspect, the disclosure provides an isolated antibody thatspecifically binds an acetaminophen-protein adduct but does notspecifically bind free acetaminophen and recognizes the immunogen:Carrier Protein-2-iminothiolane-APAP.

In another aspect, the disclosure provides an isolated antibody thatspecifically binds an acetaminophen-protein adduct but does notspecifically bind free acetaminophen and comprises a heavy chain CDR3comprising the amino acid sequence of SEQ ID NO:6 with zero to two aminoacid substitutions or SEQ ID NO:12 with zero to two amino acidsubstitutions.

In still another aspect, the disclosure provides an isolated antibodythat specifically binds acetaminophen-protein adduct but does notspecifically bind free acetaminophen and comprises a light chain CDR3comprising the amino acid sequence of SEQ ID NO:3 with zero to two aminoacid substitutions or SEQ ID NO:9 with zero to two amino acidsubstitutions.

In still yet another aspect, the disclosure provides an isolatedantibody that specifically binds acetaminophen-protein adduct but doesnot specifically bind free acetaminophen and comprises a light chainCDR3 comprising the amino acid sequence of Leu-Gly-h and/or a heavychain CDR3 comprising the amino acid sequence of SEQ ID NO:24, wherein his a hydrophobic amino acid selected from the group consisting ofalanine, valine, isoleucine, leucine, methionine, phenylalanine,tyrosine and tryptophan.

In yet another aspect, the disclosure provides a method for measuringthe amount of acetaminophen-protein adduct in a biological sample. Themethod comprises measuring the amount of acetaminophen-protein adduct ina biological sample obtained from a subject by immunoassay comprising atleast one isolated antibody that specifically bindsacetaminophen-protein adduct but does not specifically bind freeacetaminophen, wherein the antibody comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 1-12 with zero to twoamino acid substitutions or wherein the antibody specifically binds toan acetaminophen protein adduct about 2000 times more effectively thanfree acetaminophen.

In a different aspect, the disclosure provides a method for detectingacetaminophen-induced toxicity in a subject. The method comprises (i)measuring the amount of acetaminophen-protein adduct in a biologicalsample obtained from a subject by immunoassay using at least oneisolated antibody that specifically binds acetaminophen-protein adductbut does not specifically bind free acetaminophen, wherein the antibodycomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 1-12 with zero to two amino acid substitutions or wherein theantibody specifically binds to an acetaminophen protein adduct about2000 times more effectively than free acetaminophen; and (ii) comparingthe amount of acetaminophen-protein adduct in the sample to a referencevalue, wherein a greater amount of acetaminophen-protein adduct in thesample compared to the reference value indicates acetaminophen-inducedtoxicity in the subject.

In other aspects, the disclosure provides a method to determine ifhepatotoxicity in a subject is due to acetaminophen-induced toxicity.The method comprises (i) measuring the presence of and/or amount ofacetaminophen-protein adduct in a biological sample obtained from asubject by immunoassay using at least one isolated antibody thatspecifically binds acetaminophen-protein adduct but does notspecifically bind free acetaminophen, wherein the antibody comprises anamino acid sequence selected from the group consisting of SEQ ID NO:1-12 with zero to two amino acid substitutions or wherein the antibodyspecifically binds to an acetaminophen protein adduct about 2000 timesmore effectively than free acetaminophen; and (ii) determining ifacetaminophen-protein adduct is present, wherein ifacetaminophen-protein adduct is not present, the hepatotoxicity in thesubject is not due to acetaminophen-induced toxicity and wherein ifacetaminophen-protein adduct is present, comparing the amount ofacetaminophen-protein adduct in the sample to a reference value, whereina greater amount of acetaminophen-protein adduct in the sample comparedto the reference value indicates the hepatotoxicity in the subject isdue to acetaminophen-induced toxicity.

In certain aspects, the disclosure provides a method of producing amonoclonal antibody with specificity for an acetaminophen-proteinadduct. The method comprises immunizing a subject with an immunogencomprising Carrier Protein-2-iminothiolane-APAP.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1 depicts an ELISA assay showing binding of antibody 14-12 toantigen ATD-1.

FIG. 2 depicts a competitive ELISA showing the specificity of antibody14-12 for APAP-protein adducts versus free unbound APAP.

FIG. 3 depicts a competitive ELISA of RMAb clones 14-12 and 22-8. Thegraph shows the relative potency of parent drug (APAP) versus adduct(APAP-protein) as inhibitor.

FIG. 4 depicts binding of RMAb 14-12 and 22-8 to acetaminophen proteinadduct immobilized at the test band in lateral flow assays.

FIG. 5 depicts a competitive lateral flow assay with RMAb 14-12 and22-8. The graph shows the relative potency of parent drug (APAP) versusadduct (APAP-protein) as inhibitor.

FIG. 6 depicts the inhibition of APAP-protein adducts and APAP in acompetitive RMAb-based lateral flow assay. The APAP-protein adduct isphysiologically formed APAP protein adduct from the serum of an APAPtoxicity patient.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure provides antibodies that react specifically withacetaminophen-protein adducts that are formed physiologically during thepathogenesis of acetaminophen-mediated toxicity. Antibodies of theinvention do not react specifically with free acetaminophen.

The disclosure also provides methods of use of the antibodies of theinvention. Antibodies of the invention may be used to detectacetaminophen-protein adducts in a biological sample or diagnoseacetaminophen-mediated toxicity in a subject.

Additionally, the disclosure provides a novel immunogen for the purposeof preparing antibodies with specificity for acetaminophen proteinadducts. Specifically, the immunogen is Carrier Protein-2-iminothiolanelinked-acetaminophen. The novel immunogen was prepared by modifying animmunogenic carrier protein (CP) with 2-iminothiolane (2-IT) to providea highly substituted CP with numerous 5-carbon linker molecules withterminal sulfhydryl groups. This 2-IT modified CP was then covalentlymodified at the terminal sulfhydryl groups by reaction withsynthetically prepared N-acetyl-p-benzoquinone imine.

I. Antibodies

Acetaminophen (APAP)-induced toxicity is mediated by covalent binding ofthe reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) toessential proteins in the liver. At therapeutic doses, the metabolite iseffectively detoxified by conjugation with glutathione to form a3-(glutathion-S-yl)acetaminophen conjugate. After overdose, thisreaction depletes the liver of glutathione, and the metabolitecovalently binds to hepatic proteins. The major adduct formed in thisscenario is the acetaminophen-cysteine adduct,3-(cystein-S-yl)acetaminophen. Anti-acetaminophen-protein adductantibodies of the disclosure include antibodies that bind proteinadducts of acetaminophen.

As noted above, acetaminophen can form protein adducts by conjugationwith amino acids. Anti-acetaminophen-protein adduct antibodies of thedisclosure include antibodies that bind to one or moreacetaminophen-protein adducts. Specifically, an acetaminophen-proteinadduct antibody of the disclosure binds to an acetaminophen modifiedcysteine in the polypeptide chain of any adducted protein. In someembodiments, an anti-acetaminophen-protein adduct antibody binds a3-(cystein-S-yl)acetaminophen-protein adduct. In other embodiments, ananti-acetaminophen-protein adduct antibody binds a3-(glutathion-S-yl)acetaminophen-protein adduct. In differentembodiments, an anti-acetaminophen-protein adduct antibody bindsacetaminophen mercapturate. In other embodiments, ananti-acetaminophen-protein adduct antibody binds to an acetaminophenprotein adduct on a protein modified by NAPQI. Any protein with anexposed cysteine sulfhydryl is a candidate for reaction with NAPQI andresultant formation of the 3-(cystein-S-yl)acetaminophen-protein adduct.Non-limiting examples of proteins modified by NAPQI includebetaine-homocysteine S-methyltransferase 1 (BHMT), cytoplasmic aspartateaminotransferase (cAspAT), 1,4-alpha-glucan-branching enzyme,formimidoyltransferase-cyclodeaminase (FTCD), dystrophin, aldehydedehydrogenase, ATP synthase alpha-chain mitochondrial, calregulin,carbamoylphosphate synthetase I, carbonate dehydratase III (CA-III),aldehyde dehydrogenase (AHD-M1), glutamate dehydrogenase (GDH),glutamate-ammonia ligase, cellular glutathione peroxidase, glutathionetransferases (GST), glutathione S-transferase P 1, GAPDH, AdoMetsynthetase 1, macrophage 23 kDa stress protein, eIF-4A-I, 56 kDaacetaminophen-binding protein, L-iditol 2-dehydrogenase, amineN-methyltransferase, antioxidant protein 1, tropomyosin 3, urateoxidase, 10-formyltetrahydrofolate dehydrogenase, hemoglobin, 56 kDaselenium-binding protein, lamin A, cellular thyroid hormone bindingprotein, 58 kDa microsomal protein, Life Tech mouse embryo 8 5dpc10664019 Mus musculus cDNA clone, inorganic pyrophosphatase, NML Musmusculus cDNA clone, 2-4-dienoyl-CoA reductase mitochondrial,3-HAI,3-hydroxyanthranilate 3-4-dioxygenase, 94 kDa glucose-regulatedprotein, cytosolic inhibitor of Nrf2, serum albumin, and delayed earlyresponse protein 6.

In all instances, an antibody of the disclosure specifically binds oneor more acetaminophen-protein adducts but does not specifically bindfree acetaminophen. Accordingly, an antibody of the disclosure bindsacetaminophen-protein adduct more effectively than free acetaminophen.For example, an acetaminophen-protein adduct antibody binds to anacetaminophen protein about 100, about 250, about 500, about 1000, about1500, about 2000, about 2500, about 3000, about 3500, about 4000, about4500, about 5000, about 5500, about 6000, about 6500, about 7000, about7500, about 8000, about 8500, about 9000, about 9500 or about 10,000times more effectively than free acetaminophen. Additionally, anacetaminophen-protein adduct antibody binds to an acetaminophen proteinadduct about 1000 to about 2000, about 2000 to about 3000, about 3000 toabout 4000, about 4000 to about 5000, about 5000 to about 6000, about6000 to about 7000, about 7000 to about 8000, or about 8000 to about9000 times more effectively than free acetaminophen. In one embodiment,an acetaminophen-protein adduct antibody binds to an acetaminophenprotein about 2000 times more effectively than free acetaminophen. Inanother embodiment, an acetaminophen-protein adduct antibody binds to anacetaminophen protein about 8000 times more effectively than freeacetaminophen. The phrase “specifically binds” herein means antibodiesbind to the acetaminophen-protein adduct with an affinity constant orAffinity of interaction (KD) in the range of 0.1 pM to 10 nM, with apreferred range being 0.1 pM to 1 nM. Methods of determining whether anantibody binds to acetaminophen protein adducts are known in the art. Incertain embodiments, the specific antibodies may recognize anacetaminophen modified cysteine in the polypeptide chain of any adductedprotein. In some embodiments, the specific antibodies may recognize a3-(cystein-S-yl)acetaminophen-protein adduct. In other embodiments, thespecific antibodies may recognize a3-(glutathion-S-yl)acetaminophen-protein adduct. In still otherembodiments, the antibodies may recognize an acetaminophen mercapturate.

Anti-acetaminophen-protein adduct antibodies useful herein also includeall antibodies that specifically bind acetaminophen protein adducts in abiological sample. In an exemplary embodiment,anti-acetaminophen-protein adduct antibodies useful herein include allantibodies that specifically bind 3-(cystein-S-yl)acetaminophen presentin a biological sample.

In an aspect, antibodies useful herein include those antibodies whichhave been isolated, characterized, purified, are functional and havebeen recovered (obtained) for use in an assay to detectacetaminophen-protein adduct in a biological sample obtained from aliving subject and predict the development of acetaminophen toxicity inthe subject. In another aspect, antibodies useful herein include thoseantibodies which have been isolated, characterized, purified, arefunctional and have been recovered (obtained) for use in an assay todetect acetaminophen-protein adduct in a biological sample obtained froma living subject and diagnose the development of acetaminophen toxicityin the subject. In another aspect, antibodies useful herein includethose antibodies which have been isolated, characterized, purified, arefunctional and have been recovered (obtained) or for use in an assay todetect acetaminophen-protein adduct in a biological sample obtained froma living subject and classify the subject as having an increased risk ofdeveloping acetaminophen toxicity in the subject's lifetime. In anotheraspect, antibodies useful herein include those antibodies which havebeen isolated, characterized, purified, are functional and have beenrecovered (obtained) for use and are listed in Table A, as well asvariants thereof (e.g. humanized forms, chimeric forms, andimmunological fragments).

TABLE A Antibodies of the invention Antibody Name Immunogen  14-12CP-2IT-APAP 14-7 CP-2IT-APAP 22-8 CP-2IT-APAP

The term “antibody” includes the term “monoclonal antibody”. “Monoclonalantibody” refers to an antibody that is derived from a single copy orclone, including e.g., any eukaryotic, prokaryotic, or phage clone.“Monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. Monoclonal antibodies can be produced using e.g.,hybridoma techniques well known in the art, as well as recombinanttechnologies, phage display technologies, synthetic technologies orcombinations of such technologies and other technologies readily knownin the art. Furthermore, the monoclonal antibody may be labeled with adetectable label, immobilized on a solid phase and/or conjugated with aheterologous compound (e.g., an enzyme or toxin) according to methodsknown in the art.

Further by “antibody” is meant a functional monoclonal antibody, or animmunologically effective fragment thereof; such as an Fab, Fab′, orF(ab′)2 fragment thereof. In some contexts herein, fragments will bementioned specifically for emphasis; nevertheless, it will be understoodthat regardless of whether fragments are specified, the term “antibody”includes such fragments as well as single-chain forms. As long as theprotein retains the ability specifically to bind its intended target, itis included within the term “antibody.” Also included within thedefinition “antibody” for example are single chain forms, generallydesignated Fv regions, of antibodies with this specificity. Optionally,the antibodies useful in the discovery are produced recombinantly, asmanipulation of the typically rabbit or other non-human antibodies withthe appropriate specificity is required in order to convert them tohumanized form. Antibodies may or may not be glycosylated. Antibodiesare properly cross-linked via disulfide bonds, as is known.

The basic antibody unit of an antibody useful herein comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

Anti-acetaminophen-protein adduct antibodies useful herein include thosewhich are isolated, characterized, purified, function and have beenrecovered (obtained) from a process for their preparation and thusavailable for use herein in a useful form in a diagnostically sufficientamount.

Light chains are classified as kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids.

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. The chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarily determining regions (hereinafter referred to as “CDRs.”)The CDRs from the two chains are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4 respectively. The assignment of amino acids to eachdomain is in accordance with known conventions (See, Kabat “Sequences ofProteins of Immunological Interest” National Institutes of Health,Bethesda, Md., 1987 and 1991; Chothia, et al, J. Mol. Bio. (1987)196:901-917; Chothia, et al., Nature (1989) 342:878-883).

In an aspect, monoclonal anti-acetaminophen-protein adduct antibodiesare generated with appropriate specificity by standard techniques ofimmunization of mammals, forming hybridomas from the antibody-producingcells of said mammals or otherwise immortalizing them, and culturing thehybridomas or immortalized cells to assess them for the appropriatespecificity. In the present case, such antibodies could be generated byimmunizing a human, rabbit, rat or mouse, for example, with an immunogenas described in Section III. Materials for recombinant manipulation canbe obtained by retrieving the nucleotide sequences encoding the desiredantibody from the hybridoma or other cell that produces it. Thesenucleotide sequences can then be manipulated and isolated,characterized, purified and, recovered for use herein.

In an embodiment, an antibody of the invention may be humanized. As usedherein “humanized antibody” includes an anti-acetaminophen antibody thatis composed partially or fully of amino acid sequences derived from ahuman antibody germline by altering the sequence of an antibody havingnon-human complementarity determining regions (“CDR”). The simplest suchalteration may consist simply of substituting the constant region of ahuman antibody for the murine or rabbit constant region, thus resultingin a human/murine or rabbit chimera which may have sufficiently lowimmunogenicity to be acceptable for pharmaceutical use. Preferably,however, the variable region of the antibody and even the CDR is alsohumanized by techniques that are by now well known in the art. Theframework regions of the variable regions are substituted by thecorresponding human framework regions leaving the non-human CDRsubstantially intact, or even replacing the CDR with sequences derivedfrom a human genome. CDRs may also be randomly mutated such that bindingactivity and affinity for acetaminophen protein adduct is maintained orenhanced in the context of fully human germline framework regions orframework regions that are substantially human. Substantially humanframeworks have at least 90%, 95%, or 99% sequence identity with a knownhuman framework sequence. Fully useful human antibodies may also beproduced in genetically modified mice whose immune systems have beenaltered to correspond to human immune systems. As mentioned above, it issufficient for use in the methods of this discovery, to employ animmunologically specific fragment of the antibody, including fragmentsrepresenting single chain forms.

The antibodies of the present invention may also be used as fusionproteins known as single chain variable fragments (scFv). These scFvsare comprised of the heavy and light chain variable regions connected bya linker. In most instances, but not all, the linker may be a peptide. Alinker peptide is preferably from about 10 to 25 amino acids in length.Preferably, a linker peptide is rich in glycine, as well as serine orthreonine. ScFvs can be used to facilitate phage display or can be usedfor flow cytometry, immunohistochemistry, or as targeting domains.Methods of making and using scFvs are known in the art.

In a preferred embodiment, the scFvs of the present invention areconjugated to a human constant domain. In some embodiments, the heavyconstant domain is derived from an IgG domain, such as IgG1, IgG2, IgG3,or IgG4. In other embodiments, the heavy chain constant domain may bederived from IgA, IgM, or IgE.

A preferred antibody is a rabbit antibody derived from a hybridomadesignated 14-12, 14-7 or 22-8. As used herein, the term “derived from”means that the “derived” antibody comprises at least one CDR region fromthe antibody produced by 14-12, 14-7 or 22-8. Stated another way, the“derived antibody” comprises at least one CDR region comprised of theamino acid sequence selected from the group consisting of SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

In one embodiment, an antibody of the invention may be derived from thehybridoma 14-12 or 14-7, and may be encoded by a nucleic acid sequencecomprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to thelight chain variable region of SEQ ID NO:17, or may be encoded by anucleic acid sequence comprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% identity to the heavy chain variable region of SEQ ID NO:18. Inanother embodiment, an antibody of the invention may be derived from thehybridoma 14-12 or 14-7, and may be encoded by an amino acid sequencecomprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to thelight chain variable region of SEQ ID NO:13, or may be encoded by anucleic acid sequence comprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% identity to the heavy chain variable region of SEQ ID NO:14.

In a different embodiment, an antibody of the invention may be derivedfrom the hybridoma 22-8, and may be encoded by a nucleic acid sequencecomprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to thelight chain variable region of SEQ ID NO:19, or may be encoded by anucleic acid sequence comprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% identity to the heavy chain variable region of SEQ ID NO:20. Inanother embodiment, an antibody of the invention may be derived from thehybridoma 22-8, and may be encoded by an amino acid sequence comprising90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to the light chainvariable region of SEQ ID NO:15, or may be encoded by an amino acidsequence comprising 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identityto the heavy chain variable region of SEQ ID NO:16.

In an exemplary embodiment of an antibody of the invention that binds toan acetaminophen-protein adduct, the antibody comprises the light chainamino acid sequence of SEQ ID NO:13 and the heavy chain amino acidsequence of SEQ ID NO:14 [i.e. the monoclonal antibody referred toherein as 14-12 or 14-7]. In another exemplary embodiment of an antibodyof the invention that binds to an acetaminophen-protein adduct, theantibody comprises the light chain amino acid sequence of SEQ ID NO:15and the heavy chain amino acid sequence of SEQ ID NO:16 [i.e. themonoclonal antibody referred to herein as 22-8]. In another exemplaryembodiment of an antibody of the invention that binds to anacetaminophen-protein adduct, the antibody comprises the light chainnucleic acid sequence of SEQ ID NO:17 and the heavy chain amino acidsequence of SEQ ID NO:18 [i.e. the monoclonal antibody referred toherein as mAb 14-12 or 14-7]. In another exemplary embodiment of anantibody of the invention that binds to an acetaminophen-protein adduct,the antibody comprises the light chain nucleic acid sequence of SEQ IDNO:19 and the heavy chain nucleic acid sequence of SEQ ID NO:20 [i.e.the monoclonal antibody referred to herein as 22-8].

In one embodiment, an antibody of the invention may comprise a lightchain CDR1, such as the antibodies 1, 49 and 97 of Table B. In anotherembodiment, an antibody of the invention may comprise a light chainCDR2, such as the antibodies 4, 52 and 100 of Table B. In yet anotherembodiment, an antibody of the invention may comprise a light chainCDR3, such as the antibodies 6, 54 and 102 of Table B. In an alternativeembodiment, an antibody of the invention may comprise a combination oftwo or three light chain CDRs, such as the antibodies 2, 3, 5, 50, 51,53, 98, 99 and 101 of Table B.

Similarly, in one embodiment, an antibody of the invention may comprisea heavy chain CDR1, such as the antibodies 7, 55 and 103 of Table B. Inanother embodiment, an antibody of the invention may comprise a heavychain CDR2, such as the antibodies 10, 58 and 106 of Table B. In yetanother embodiment, an antibody of the invention may comprise a heavychain CDR3, such as the antibodies 12, 60 and 108 of Table B. In analternative embodiment, an antibody of the invention may comprise acombination of two or three heavy chain CDRs, such as the antibodies 8,9, 11, 56, 57, 59, 104, 105 and 107 of Table B.

Alternatively, an antibody of the invention may comprise one or morelight chain CDRs and one or more heavy chain CDRs, such as theantibodies 13-48, 61-96 and 109-144 of Table B.

TABLE B Anti- Light Chain Heavy Chain body CDR1 CDR2 CDR3 CDR1 CDR2 CDR31 SEQ ID NO: 1 2 SEQ ID NO: 1 SEQ ID NO: 2 3 SEQ ID NO: 1 SEQ ID NO: 2SEQ ID NO: 3 4 SEQ ID NO: 2 5 SEQ ID NO: 2 SEQ ID NO: 3 6 SEQ ID NO: 3 7SEQ ID NO: 4 8 SEQ ID NO: 4 SEQ ID NO: 5 9 SEQ ID NO: 4 SEQ ID NO: 5 SEQID NO: 6 10 SEQ ID NO: 5 11 SEQ ID NO: 5 SEQ ID NO: 6 12 SEQ ID NO: 6 13SEQ ID NO: 1 SEQ ID NO: 4 14 SEQ ID NO: 1 SEQ ID NO: 4 SEQ ID NO: 5 15SEQ ID NO: 1 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 16 SEQ ID NO: 1 SEQID NO: 5 17 SEQ ID NO: 1 SEQ ID NO: 5 SEQ ID NO: 6 18 SEQ ID NO: 1 SEQID NO: 6 19 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 4 20 SEQ ID NO: 1 SEQID NO: 2 SEQ ID NO: 4 SEQ ID NO: 5 21 SEQ ID NO: 1 SEQ ID NO: 2 SEQ IDNO: 4 SEQ ID NO: 5 SEQ ID NO: 6 22 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO:5 23 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 5 SEQ ID NO: 6 24 SEQ ID NO: 1SEQ ID NO: 2 SEQ ID NO: 6 25 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQID NO: 4 26 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ IDNO: 5 27 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO:5 SEQ ID NO: 6 28 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 5 29SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 5 SEQ ID NO: 6 30 SEQID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 6 31 SEQ ID NO: 2 SEQ IDNO: 4 32 SEQ ID NO: 2 SEQ ID NO: 4 SEQ ID NO: 5 33 SEQ ID NO: 2 SEQ IDNO: 4 SEQ ID NO: 5 SEQ ID NO: 6 34 SEQ ID NO: 2 SEQ ID NO: 5 35 SEQ IDNO: 2 SEQ ID NO: 5 SEQ ID NO: 6 36 SEQ ID NO: 2 SEQ ID NO: 6 37 SEQ IDNO: 2 SEQ ID NO: 3 SEQ ID NO: 4 38 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO:4 SEQ ID NO: 5 39 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5SEQ ID NO: 6 40 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 5 41 SEQ ID NO: 2SEQ ID NO: 3 SEQ ID NO: 5 SEQ ID NO: 6 42 SEQ ID NO: 2 SEQ ID NO: 3 SEQID NO: 6 43 SEQ ID NO: 3 SEQ ID NO: 4 44 SEQ ID NO: 3 SEQ ID NO: 4 SEQID NO: 5 45 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 46 SEQID NO: 3 SEQ ID NO: 5 47 SEQ ID NO: 3 SEQ ID NO: 5 SEQ ID NO: 6 48 SEQID NO: 3 SEQ ID NO: 6 49 SEQ ID NO: 7 50 SEQ ID NO: 7 SEQ ID NO: 8 51SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 52 SEQ ID NO: 8 53 SEQ ID NO: 8SEQ ID NO: 9 54 SEQ ID NO: 9 55 SEQ ID NO: 10 56 SEQ ID NO: 10 SEQ IDNO: 11 57 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 58 SEQ ID NO: 11 59SEQ ID NO: 11 SEQ ID NO: 12 60 SEQ ID NO: 12 61 SEQ ID NO: 7 SEQ ID NO:10 62 SEQ ID NO: 7 SEQ ID NO: 10 SEQ ID NO: 11 63 SEQ ID NO: 7 SEQ IDNO: 10 SEQ ID NO: 11 SEQ ID NO: 12 64 SEQ ID NO: 7 SEQ ID NO: 11 65 SEQID NO: 7 SEQ ID NO: 11 SEQ ID NO: 12 66 SEQ ID NO: 7 SEQ ID NO: 12 67SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 10 68 SEQ ID NO: 7 SEQ ID NO: 8 SEQID NO: 10 SEQ ID NO: 11 69 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 10 SEQID NO: 11 SEQ ID NO: 12 70 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 11 71SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 11 SEQ ID NO: 12 72 SEQ ID NO: 7SEQ ID NO: 8 SEQ ID NO: 12 73 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQID NO: 10 74 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ IDNO: 11 75 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 SEQ IDNO: 11 SEQ ID NO: 12 76 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ IDNO: 11 77 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 11 SEQ IDNO: 12 78 SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 12 79 SEQ IDNO: 8 SEQ ID NO: 10 80 SEQ ID NO: 8 SEQ ID NO: 10 SEQ ID NO: 11 81 SEQID NO: 8 SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 82 SEQ ID NO: 8 SEQID NO: 11 83 SEQ ID NO: 8 SEQ ID NO: 11 SEQ ID NO: 12 84 SEQ ID NO: 8SEQ ID NO: 12 85 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 86 SEQ ID NO: 8SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 87 SEQ ID NO: 8 SEQ ID NO: 9SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 88 SEQ ID NO: 8 SEQ ID NO: 9SEQ ID NO: 11 89 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 11 SEQ ID NO: 1290 SEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 12 91 SEQ ID NO: 9 SEQ ID NO: 1092 SEQ ID NO: 9 SEQ ID NO: 10 SEQ ID NO: 11 93 SEQ ID NO: 9 SEQ ID NO:10 SEQ ID NO: 11 SEQ ID NO: 12 94 SEQ ID NO: 9 SEQ ID NO: 11 95 SEQ IDNO: 9 SEQ ID NO: 11 SEQ ID NO: 12 96 SEQ ID NO: 9 SEQ ID NO: 12 97 SEQID NO: 21 98 SEQ ID NO: 21 SEQ ID NO: 22 99 SEQ ID NO: 21 SEQ ID NO: 22Leu-Gly-h* 100 SEQ ID NO: 22 101 SEQ ID NO: 22 Leu-Gly-h* 102 Leu-Gly-h*103 Tyr-X-Ile** 104 Tyr-X-Ile** SEQ ID NO: 23 105 Tyr-X-Ile** SEQ ID NO:23 SEQ ID NO: 24 106 SEQ ID NO: 23 107 SEQ ID NO: 23 SEQ ID NO: 24 108SEQ ID NO: 24 109 SEQ ID NO: 21 Tyr-X-Ile** 110 SEQ ID NO: 21Tyr-X-Ile** SEQ ID NO: 23 111 SEQ ID NO: 21 Tyr-X-Ile** SEQ ID NO: 23SEQ ID NO: 24 112 SEQ ID NO: 21 SEQ ID NO: 23 113 SEQ ID NO: 21 SEQ IDNO: 23 SEQ ID NO: 24 114 SEQ ID NO: 21 SEQ ID NO: 24 115 SEQ ID NO: 21SEQ ID NO: 22 Tyr-X-Ile** 116 SEQ ID NO: 21 SEQ ID NO: 22 Tyr-X-Ile**SEQ ID NO: 23 117 SEQ ID NO: 21 SEQ ID NO: 22 Tyr-X-Ile** SEQ ID NO: 23SEQ ID NO: 24 118 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23 119 SEQ IDNO: 21 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 120 SEQ ID NO: 21 SEQID NO: 22 SEQ ID NO: 24 121 SEQ ID NO: 21 SEQ ID NO: 22 Leu-Gly-h*Tyr-X-Ile** 122 SEQ ID NO: 21 SEQ ID NO: 22 Leu-Gly-h* Tyr-X-Ile** SEQID NO: 23 123 SEQ ID NO: 21 SEQ ID NO: 22 Leu-Gly-h* Tyr-X-Ile** SEQ IDNO: 23 SEQ ID NO: 24 124 SEQ ID NO: 21 SEQ ID NO: 22 Leu-Gly-h* SEQ IDNO: 23 125 SEQ ID NO: 21 SEQ ID NO: 22 Leu-Gly-h* SEQ ID NO: 23 SEQ IDNO: 24 126 SEQ ID NO: 21 SEQ ID NO: 22 Leu-Gly-h* SEQ ID NO: 24 127 SEQID NO: 22 Tyr-X-Ile** 128 SEQ ID NO: 22 Tyr-X-Ile** SEQ ID NO: 23 129SEQ ID NO: 22 Tyr-X-Ile** SEQ ID NO: 23 SEQ ID NO: 24 130 SEQ ID NO: 22SEQ ID NO: 23 131 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 132 SEQ IDNO: 22 SEQ ID NO: 24 133 SEQ ID NO: 22 Leu-Gly-h* Tyr-X-Ile** 134 SEQ IDNO: 22 Leu-Gly-h* Tyr-X-Ile** SEQ ID NO: 23 135 SEQ ID NO: 22 Leu-Gly-h*Tyr-X-Ile** SEQ ID NO: 23 SEQ ID NO: 24 136 SEQ ID NO: 22 Leu-Gly-h* SEQID NO: 23 137 SEQ ID NO: 22 Leu-Gly-h* SEQ ID NO: 23 SEQ ID NO: 24 138SEQ ID NO: 22 Leu-Gly-h* SEQ ID NO: 24 139 Leu-Gly-h* Tyr-X-Ile** 140Leu-Gly-h* Tyr-X-Ile** SEQ ID NO: 23 141 Leu-Gly-h* Tyr-X-Ile** SEQ IDNO: 23 SEQ ID NO: 24 142 Leu-Gly-h* SEQ ID NO: 23 143 Leu-Gly-h* SEQ IDNO: 23 SEQ ID NO: 24 144 Leu-Gly-h* SEQ ID NO: 24 *wherein h is ahydrophobic amino acid selected from the group consisting of alanine,valine, isoleucine, leucine, methionine, phenylalanine, tyrosine andtryptophan. *wherein X is any amino acid.

In one embodiment, an antibody of the invention may comprise a lightchain variable region comprising a CDR1 of amino acid sequence SEQ IDNO: 1 with zero to two amino acid substitutions, a CDR2 of amino acidsequence SEQ ID NO: 2 with zero to two amino acid substitutions, and aCDR3 of amino acid sequence SEQ ID NO:3 with zero to two amino acidsubstitutions, or may comprise a heavy chain variable region comprisinga CDR1 of amino acid sequence SEQ ID NO: 4 with zero to two amino acidsubstitutions, a CDR2 of amino acid sequence SEQ ID NO: 5 with zero totwo amino acid substitutions, and a CDR3 of amino acid sequence SEQ IDNO: 6 with zero to two amino acid substitutions. In a preferredembodiment, an antibody of the invention may comprise a light chainvariable region comprising a CDR1 of amino acid sequence SEQ ID NO: 1with zero to two amino acid substitutions, a CDR2 of amino acid sequenceSEQ ID NO: 2 with zero to two amino acid substitutions, a CDR3 of aminoacid sequence SEQ ID NO:3, a heavy chain variable region comprising aCDR1 of amino acid sequence SEQ ID NO: 4 with zero to two amino acidsubstitutions, a CDR2 of amino acid sequence SEQ ID NO: 5 with zero totwo amino acid substitutions, and a CDR3 of amino acid sequence SEQ IDNO: 6 with zero to two amino acid substitutions. In an exemplaryembodiment, an antibody of the invention may comprise a light chainvariable region comprising a CDR1 of amino acid sequence SEQ ID NO: 1, aCDR2 of amino acid sequence SEQ ID NO: 2, a CDR3 of amino acid sequenceSEQ ID NO:3, a heavy chain variable region comprising a CDR1 of aminoacid sequence SEQ ID NO: 4, a CDR2 of amino acid sequence SEQ ID NO: 5,and a CDR3 of amino acid sequence SEQ ID NO: 6. The invention alsoencompasses the corresponding nucleic acid sequences of SEQ ID NO:1, 2,3, 4, 5, and 6, which can readily be determined by one of skill in theart, and may be incorporated into a vector or other large DNA molecule,such as a chromosome, in order to express an antibody of the invention.

In another embodiment, an antibody of the invention may comprise a lightchain variable region comprising a CDR1 of amino acid sequence SEQ IDNO: 7 with zero to two amino acid substitutions, a CDR2 of amino acidsequence SEQ ID NO: 8 with zero to two amino acid substitutions, and aCDR3 of amino acid sequence SEQ ID NO: 9 with zero to two amino acidsubstitutions, or may comprise a heavy chain variable region comprisinga CDR1 of amino acid sequence SEQ ID NO: 10 with zero to two amino acidsubstitutions, a CDR2 of amino acid sequence SEQ ID NO: 11 with zero totwo amino acid substitutions, and a CDR3 of amino acid sequence SEQ IDNO: 12 with zero to two amino acid substitutions. In a preferredembodiment, an antibody of the invention may comprise a light chainvariable region comprising a CDR1 of amino acid sequence SEQ ID NO: 7with zero to two amino acid substitutions, a CDR2 of amino acid sequenceSEQ ID NO: 8 with zero to two amino acid substitutions, a CDR3 of aminoacid sequence SEQ ID NO: 9 with zero to two amino acid substitutions,and a heavy chain variable region comprising a CDR1 of amino acidsequence SEQ ID NO: 10 with zero to two amino acid substitutions, a CDR2of amino acid sequence SEQ ID NO: 11 with zero to two amino acidsubstitutions, and a CDR3 of amino acid sequence SEQ ID NO: 12 with zeroto two amino acid substitutions. In an exemplary embodiment, an antibodyof the invention may comprise a light chain variable region comprising aCDR1 of amino acid sequence SEQ ID NO: 7, a CDR2 of amino acid sequenceSEQ ID NO: 8, a CDR3 of amino acid sequence SEQ ID NO: 9, a heavy chainvariable region comprising a CDR1 of amino acid sequence SEQ ID NO: 10,a CDR2 of amino acid sequence SEQ ID NO: 11, and a CDR3 of amino acidsequence SEQ ID NO: 12. The invention also encompasses the correspondingnucleic acid sequences of SEQ ID NO: 7, 8, 9, 10, 11, and 12, which canreadily be determined by one of skill in the art, and may beincorporated into a vector or other large DNA molecule, such as achromosome, in order to express an antibody of the invention.

In one embodiment, an antibody of the invention may comprise a lightchain variable region comprising a CDR1 of amino acid sequence SEQ IDNO: 21 (QXSQphXR, wherein X is any amino acid, p is a polar amino acidand h is a hydrophobic amino acid), a CDR2 of amino acid sequence SEQ IDNO: 22 (XhXpLXS, wherein X is any amino acid, p is a polar amino acidand h is a hydrophobic amino acid), and/or a CDR3 of amino acid sequenceLeu-Gly-h (wherein h is a hydrophobic residue), or may comprise a heavychain variable region comprising a CDR1 of amino acid sequence Tyr-X-Ile(wherein X is any amino acid), a CDR2 of amino acid sequence SEQ ID NO:23 (AXYAXWXKG, wherein X is any amino acid), and/or a CDR3 of amino acidsequence SEQ ID NO: 24 (hXXGGhhXX, wherein X is any amino acid and h isa hydrophobic amino acid). In another embodiment, an antibody of theinvention may comprise a light chain variable region comprising a CDR1of amino acid sequence SEQ ID NO: 21 (QXSQphXR, wherein X is any aminoacid, p is a polar amino acid and h is a hydrophobic amino acid), a CDR2of amino acid sequence SEQ ID NO: 22 (XhXpLXS, wherein X is any aminoacid, p is a polar amino acid and h is a hydrophobic amino acid), and aCDR3 of amino acid sequence Leu-Gly-h (wherein h is a hydrophobicresidue), and may comprise a heavy chain variable region comprising aCDR1 of amino acid sequence Tyr-X-Ile (wherein X is any amino acid), aCDR2 of amino acid sequence SEQ ID NO: 23 (AXYAXWXKG, wherein X is anyamino acid), and a CDR3 of amino acid sequence SEQ ID NO: 24 (hXXGGhhXX,wherein X is any amino acid and h is a hydrophobic amino acid). In stillanother embodiment, an antibody of the invention may comprise a lightchain variable region comprising a CDR3 of amino acid sequence Leu-Gly-h(wherein h is a hydrophobic residue), and/or may comprise a heavy chainvariable region comprising a CDR3 of amino acid sequence SEQ ID NO: 24(hXXGGhhXX, wherein X is any amino acid and h is a hydrophobic aminoacid). In each of the foregoing embodiments, SEQ ID NO: 21 may furthercomprise 1, 2, 3 or 4 amino acids on the C-terminus; Leu-Gly-h mayfurther comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on theC-terminus; Tyr-X-Ile may further comprise 1 amino acid on theC-terminus and/or 1 amino acid on the N-terminus; SEQ ID NO: 23 mayfurther comprise 1, 2, 3, 4, 5, 6 or 7 amino acids on the N-terminus;SEQ ID NO: 24 may further comprise 1 amino acid on the C-terminus.

As used herein, a polar amino acid is selected from the group consistingof serine, threonine, asparagine, and glutamine and a hydrophobic aminoacid is selected from the group consisting of alanine, valine,isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

TABLE C Sequence Listing SEQ ID NO: Description SEQUENCE  1mAb 14-12 and 14-7 QASQSISRQVS LC CDR1  2 mAb 14-12 and 14-7 RASTLASLC CDR2  3 mAb 14-12 and 14-7 LGIVTDRIADGLA LC CDR3  4mAb 14-12 and 14-7 AYGIN HC CDR1  5 mAb 14-12 and 14-7 FSAPHTASYARWTKGHC CDR2  6 mAb 14-12 and 14-7 YDRGGMVFNL HC CDR3  7 mAb 22-8 LC CDR1QSSQNVFRKNYLS  8 mAb 22-8 LC CDR2 YIDSLTS  9 mAb 22-8 LC CDR3LGVDGSANDAT 10 mAb 22-8 HC CDR1 NYYII 11 mAb 22-8 HC CDR2ITYGGGFAYYASWAKG 12 mAb 22-8 HC CDR3 AAAGGAYDL 13 mAb 14-12 and 14-7MDTRAPTQLLGLLLLWLPGATFALVMTQTPSSVPAAVGGTVTIGCQA LC AA sequenceSQSISRQVSWYQQKPGQPPKLLIYRASTLASGVSSRFKGSGSGTEFTLTISGVQCDDAATYYCLGIVTDRIADGLAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC 14 mAb 14-12 and 14-7METGLRWLLLVAVLKGVQCQSVEESGGRLVTPGTPLTLTCTVSGFSI HC AA sequenceNAYGINWVRQAPGKGLEYIGFSAPHTASYARWTKGRFTMSRTSTTVDLRMTSPTTEDTATYFCARYDRGGMVFNLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK 15 mAb 22-8 LC AAMDTRAPTQLLGLLLLWLPGATFAIEMTQTPSPVSAVVGGTVTINCQSS sequenceQNVFRKNYLSWFQQKPGQPPKLLISYIDSLTSGVPSRFSGSGAGTQFTLTISDVQCDDAATYYCLGVDGSANDATFGGGTEVVVEGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC 16 mAb 22-8 HC variableMETGLRWLLLVAVLKGVQCQSLEESGGRLVTPGGSLTLTCTASGLTI domainNNYYIIWVRQAPGKGLKYIGITYGGGFAYYASWAKGRFTISRTSTTVDLKMTSLTAEDTATYFCVRAAAGGAYDLWGQGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK 17 mAb 14-12 and 14-7ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCT LC NT sequenceCTGGCTCCCAGGTGCCACATTTGCCCTGGTGATGACCCAGACTCCATCCTCCGTGCCTGCCGCTGTGGGAGGCACAGTCACCATCGGTTGCCAGGCCAGTCAGAGTATTAGTAGGCAAGTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAGGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACTATTAGCGGCGTCCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTATTGTTACTGACCGTATTGCTGATGGGCTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG 18 mAb 14-12 and 14-7ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAA HC NT sequenceGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGCCTAGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGATTCAGCATCAATGCCTATGGAATTAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGATTCAGTGCTCCTCATACCGCATCCTACGCGAGGTGGACAAAGGGCCGATTCACCATGTCCAGAACCTCGACCACGGTGGATCTGAGAATGACCAGCCCAACAACCGAGGACACGGCCACCTACTTTTGTGCCAGATATGATCGGGGTGGGATGGTATTTAACTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 19 mAb 22-8 LC NTATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCT sequenceCTGGCTCCCAGGTGCCACATTTGCCATTGAAATGACCCAGACTCCATCCCCTGTGTCTGCAGTTGTGGGAGGCACAGTCACCATCAATTGTCAGTCCAGTCAGAACGTTTTTCGTAAGAACTATTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCAGTTATATAGACAGTCTGACATCTGGGGTCCCATCGCGATTCAGCGGCAGTGGAGCTGGGACACAGTTCACTCTCACCATCAGTGACGTGCAGTGTGACGATGCTGCCACTTATTACTGTTTAGGCGTTGATGGTAGTGCTAATGATGCTACTTTCGGCGGAGGGACCGAGGTGGTGGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG 20 mAb 22-8 HC NTATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAA sequenceGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGAGGATCCCTGACACTCACCTGCACAGCCTCTGGACTCACCATCAATAACTACTACATAATTTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGAAATACATCGGAATCACCTATGGTGGTGGTTTTGCATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAACCTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAGCCGAGGACACGGCCACTTATTTCTGTGTCAGAGCTGCGGCTGGTGGTGCTTATGATTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 21 LC CDR1QXSQphXR, wherein X is any amino acid, p is a polaramino acid and h is a hydrophobic amino acid 22 LC CDR2XhXpLXS; wherein X is any amino acid, p is a polaramino acid and h is a hydrophobic amino acid LC CDR3LGh; wherein h is a hydrophobic amino acid HC CDR1YXI, wherein X is any amino acid 23 HC CDR2AXYAXWXKG, wherein X is any amino acid 24 HC CDR3hXXGGhhXX, wherein X is any amino acid and h is a hydrophobic amino acidII. Methods of Using Anti-Acetaminophen-Protein Adduct Antibodies

In an aspect, the present disclosure provides antibodies to detectacetaminophen-protein adducts in a biological sample obtained from asubject. In another aspect, the present disclosure provides antibodiesto measure the amount of acetaminophen-protein adducts in a biologicalsample obtained from a subject. The amount of acetaminophen-proteinadducts in a biological sample obtained from a subject can be used toclassify a subject as having high or low amounts ofacetaminophen-protein adducts, and may be further used to identify inthe subject exposure and/or toxicity associated with acetaminophen. In aspecific embodiment, the acetaminophen-protein adduct is3-(cystein-S-yl)acetaminophen-protein adduct.

(a) Methods to Detect and Measure the Amount of Acetaminophen-ProteinAdduct in a Biological Sample

In an aspect, the disclosure provides means to detectacetaminophen-protein adduct in a biological sample obtained from asubject. In another aspect, the disclosure provides means to measure theamount of acetaminophen-protein adduct in a biological sample obtainedfrom a subject. The method generally comprises detecting and/ormeasuring the amount of one or more acetaminophen-protein adduct in abiological sample obtained from a subject using an antibody thatspecifically binds acetaminophen-protein adduct. Additionally, themethod may comprise (i) obtaining a biological sample from a subject,and (ii) detecting and/or measuring the amount of one or moreacetaminophen-protein adduct in the sample using an antibody thatspecifically binds acetaminophen-protein adduct. Suitable antibodies aredescribed above in Section I.

As used herein, the term “subject” refers to a living organism that maybe administered acetaminophen. Suitable subjects include, but are notlimited to, a human, a livestock animal, a companion animal, a labanimal, and a zoological animal. In one embodiment, the subject may be arodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment,the subject may be a livestock animal. Non-limiting examples of suitablelivestock animals may include pigs, cows, horses, goats, sheep, llamasand alpacas. In yet another embodiment, the subject may be a companionanimal. Non-limiting examples of companion animals may include pets suchas dogs, cats, rabbits, and birds. In yet another embodiment, thesubject may be a zoological animal. As used herein, a “zoologicalanimal” refers to an animal that may be found in a zoo. Such animals mayinclude non-human primates, large cats, wolves, and bears. In specificembodiments, the animal is a laboratory animal. Non-limiting examples ofa laboratory animal may include rodents, canines, felines, and non-humanprimates. In certain embodiments, the animal is a rodent. Non-limitingexamples of rodents may include mice, rats, guinea pigs, etc. In apreferred embodiment, the subject is human. Subjects may be of any age,including newborn, adolescent, adult, middle age, or elderly.

A subject may or may not be having a symptom associated withacetaminophen-induced toxicity. Specifically, the acetaminophen-inducedtoxicity may be hepatotoxicity. A skilled artisan will appreciate thatpathological acetaminophen-induced toxicity likely commences prior todiagnosis or the onset of symptoms associated with acetaminophen-inducedtoxicity. In some embodiments, a subject is having a symptom associatedwith acetaminophen-induced toxicity. In other embodiments, a subject isnot having a symptom associated with acetaminophen-induced toxicity. Instill other embodiments, a subject has detectable acetaminophen-inducedtoxicity but is not having any other symptom associated withacetaminophen-induced toxicity. In yet still other embodiments, asubject has received acetaminophen. In different embodiments, a subjecthas received a supratherapeutic dose of acetaminophen. In alternativeembodiments, a subject has been suspected of receiving asupratherapeutic dose of acetaminophen. For example, a subject may haveliver failure of unclear etiology which may have developed as a resultof receiving a supratherapeutic dose of acetaminophen. Early diagnosisof acetaminophen-induced toxicity in the subject may reduce thedevelopment and/or progression of symptoms associated with thepathological acetaminophen-induced toxicity.

Exemplary symptoms associated with acetaminophen-induced hepatotoxicitymay include, but is not limited to, anorexia, nausea, vomiting, rightupper quadrant abdominal pain, elevated AST, ALT, bilirubin and PT(INR), renal failure, pancreatitis, multiple organ failure. Mildacetaminophen poisoning may not cause symptoms, and when present,symptoms are usually minor until 48 h after ingestion. In someembodiments, the severity of symptoms of acetaminophen toxicity arequantified using 4 stages as shown in Table D.

TABLE D Stages of acute acetaminophen poisoning Time Stage PostingestionDescription I  0-24 h Anorexia, nausea, vomiting II 24-72 h Right upperquadrant abdominal pain (common) AST, ALT, and, if poisoning is severe,bilirubin and PT (INR) sometimes elevated III 72-96 h Vomiting andsymptoms of liver failure Peaking of AST, ALT, bilirubin and INRSometimes renal failure and pancreatitis IV   >5 days Resolution ofhepatotoxicity or progression to multiple organ failure (sometimesfatal)

As used herein, the term “biological sample” refers to a sample obtainedfrom a subject. Any biological sample comprising anacetaminophen-protein adduct is suitable. Numerous types of biologicalsamples are known in the art. Suitable biological samples may include,but are not limited to, hair, tissue samples or bodily fluids. In someembodiments, the biological sample is a tissue sample such as a tissuebiopsy. The tissue biopsy may be a biopsy of liver tissue. The biopsiedtissue may be fixed, embedded in paraffin or plastic, and sectioned, orthe biopsied tissue may be frozen and cryosectioned. Alternatively, thebiopsied tissue may be processed into individual cells or an explant, orprocessed into a homogenate, a cell extract, a membranous fraction, or aprotein extract. In other embodiments, the sample may be a bodily fluid.Non-limiting examples of suitable bodily fluids include blood, plasma,serum, urine, saliva, semen, perspiration, tears, mucus, sputum, tissuelystates or other excrement (e.g. feces). In a specific embodiment, thebodily fluid is urine. In another specific embodiment, the bodily fluidis plasma. In still another specific embodiment, the bodily fluid isserum. In yet still another specific embodiment, the bodily fluid issaliva. The fluid may be used “as is”, the cellular components may beisolated from the fluid, or a protein fraction may be isolated from thefluid using standard techniques. In a different embodiment, thebiological sample is hair.

As will be appreciated by a skilled artisan, the method of collecting abiological sample can and will vary depending upon the nature of thebiological sample and the type of analysis to be performed. Any of avariety of methods generally known in the art may be utilized to collecta biological sample. Generally speaking, the method preferably maintainsthe integrity of the sample such that an acetaminophen-protein adductcan be accurately detected and the amount measured according to theinvention.

In some embodiments, a single sample is obtained from a subject todetect an acetaminophen-protein adduct in the sample. Alternatively, anacetaminophen-protein adduct may be detected in samples obtained overtime from a subject. As such, more than one sample may be collected froma subject over time. For instance, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or more samples may be collected from a subject overtime. In some embodiments, 2, 3, 4, 5, or 6 samples are collected from asubject over time. In other embodiments, 6, 7, 8, 9, or 10 samples arecollected from a subject over time. In yet other embodiments, 10, 11,12, 13, or 14 samples are collected from a subject over time. In otherembodiments, 14, 15, 16 or more samples are collected from a subjectover time.

When more than one sample is collected from a subject over time, samplesmay be collected every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ormore hours. In some embodiments, samples are collected every 0.5, 1, 2,3, or 4 hours. In other embodiments, samples are collected every 4, 5,6, or 7 hours. In yet other embodiments, samples are collected every 7,8, 9, or 10 hours. In other embodiments, samples are collected every 10,11, 12 or more hours. Additionally, samples may be collected every 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days. In some embodiments, asample is collected about every 6 days. In some embodiments, samples arecollected every 1, 2, 3, 4, or 5 days. In other embodiments, samples arecollected every 5, 6, 7, 8, or 9 days. In yet other embodiments, samplesare collected every 9, 10, 11, 12 or more days.

Once a sample is obtained, it is processed in vitro in order to detectand measure the amount of one or more acetaminophen-protein adduct usingan anti-acetaminophen-protein adduct antibody. All suitable methods fordetecting and measuring an amount of protein using an antibody known toone of skill in the art are contemplated within the scope of theinvention. Methods for detecting and measuring an amount of proteinusing an antibody (i.e. “antibody-based methods”) are well known in theart. Non-limiting examples include an ELISA, a lateral flow assay, asandwich immunoassay, a radioimmunoassay, an immunoblot or Western blot,flow cytometry, immunohistochemistry, and an array. A lateral flow assaymay be a device intended to detect the presence (or absence) of a targetanalyte in sample.

In general, an antibody-based method of detecting and measuring anamount of an acetaminophen-protein adduct comprises contacting some orall of the sample comprising an acetaminophen-protein adduct with ananti-acetaminophen-protein adduct antibody under conditions effective toallow for formation of a complex between the antibody and theacetaminophen-protein adduct. Typically, the entire sample is notneeded, allowing one skilled in the art to repeatedly detect and measurethe amount of an acetaminophen-protein adduct in the sample over time.The method may occur in solution, or the antibody oracetaminophen-protein adduct may be immobilized on a solid surface.Non-limiting examples of suitable surfaces include microtitre plates,test tubes, beads, resins, and other polymers. Attachment to thesubstrate may occur in a wide variety of ways, as will be appreciated bythose in the art. For example, the substrate and the antibody may bederivatized with chemical functional groups for subsequent attachment ofthe two. For example, the substrate may be derivatized with a chemicalfunctional group including, but not limited to, amino groups, carboxylgroups, oxo groups or thiol groups. Using these functional groups, theantibody may be attached directly using the functional groups orindirectly using linkers. An anti-acetaminophen-protein adduct antibodymay also be attached to the substrate non-covalently. For example, abiotinylated anti-acetaminophen-protein adduct antibody may be prepared,which may bind to surfaces covalently coated with streptavidin,resulting in attachment. Alternatively, an antibody may be synthesizedon the surface using techniques such as photopolymerization andphotolithography.

Contacting the sample with an antibody under effective conditions for aperiod of time sufficient to allow formation of a complex generallyinvolves adding the anti-acetaminophen-protein adduct antibodycomposition to the sample and incubating the mixture for a period oftime long enough for the anti-acetaminophen-protein adduct antibody tobind to any antigen present. After this time, the complex may be washedand then the complex is detected and the amount measured by any methodwell known in the art. Methods of detecting and measuring an amount ofan antibody-polypeptide complex are generally based on the detection ofa label or marker. The term “label”, as used herein, refers to anysubstance attached to an antibody, or other substrate material, in whichthe substance is detectable by a detection method. Non-limiting examplesof suitable labels include luminescent molecules, chemiluminescentmolecules, fluorochromes, fluorescent quenching agents, coloredmolecules, radioisotopes, scintillants, biotin, avidin, stretpavidin,protein A, protein G, antibodies or fragments thereof, polyhistidine,Ni²⁺, Flag tags, myc tags, heavy metals, and enzymes (including alkalinephosphatase, peroxidase, glucose oxidase and luciferase). Methods ofdetecting and measuring an amount of an antibody-polypeptide complexbased on the detection of a label or marker are well known in the art.

In some embodiments, an antibody-based method is an immunoassay.Immunoassays can be run in a number of different formats. Generallyspeaking, immunoassays can be divided into two categories: competitiveimmmunoassays and non-competitive immunoassays. In a competitiveimmunoassay, an unlabeled analyte in a sample competes with labeledanalyte to bind an antibody. Unbound analyte is washed away and thebound analyte is measured. In a non-competitive immunoassay, theantibody is labeled, not the analyte. Non-competitive immunoassays mayuse one antibody (e.g. the capture antibody is labeled) or more than oneantibody (e.g. at least one capture antibody which is unlabeled and atleast one “capping” or detection antibody which is labeled). Suitablelabels are described above.

In other embodiments, an antibody-based method is an immunoblot orWestern blot. In yet other embodiments, an antibody-based method is flowcytometry. In different embodiments, an antibody-based method isimmunohistochemistry (IHC). IHC uses an antibody to detect and quantifyantigens in intact tissue samples. The tissue samples may befresh-frozen and/or formalin-fixed, paraffin-embedded (orplastic-embedded) tissue blocks prepared for study by IHC. Methods ofpreparing tissue block for study by IHC, as well as methods ofperforming IHC are well known in the art.

In alternative embodiments, an antibody-based method is an array. Anarray comprises at least one address, wherein at least one address ofthe array has disposed thereon an anti-acetaminophen-protein adductantibody. Arrays may comprise from about 1 to about several hundredthousand addresses. Several substrates suitable for the construction ofarrays are known in the art, and one skilled in the art will appreciatethat other substrates may become available as the art progresses.Suitable substrates are also described above. In some embodiments, thearray comprises at least one anti-acetaminophen-protein adduct antibodyattached to the substrate is located at one or more spatially definedaddresses of the array. For example, an array may comprise at least one,at least two, at least three, at least four, or at least fiveanti-acetaminophen-protein adduct antibodies, each antibody recognizingthe same or different acetaminophen-protein adducts, and each antibodymay be may be at one, two, three, four, five, six, seven, eight, nine,ten or more spatially defined addresses.

For each of the foregoing embodiments, an acetaminophen-protein adductmay be first isolated or enriched before detection. For instance, anacetaminophen-protein adduct may be enriched or isolated using liquidchromatography, by precipitation, electrophoresis, or affinitypurification. In some embodiments, an acetaminophen-protein adduct maybe enriched or purified using liquid chromatography. In otherembodiments, an acetaminophen-protein adduct may be enriched or purifiedusing electrophoresis.

In an embodiment, an acetaminophen-protein adduct may be enriched orpurified by affinity purification before detection. In anotherembodiment, an acetaminophen-protein adduct may be enriched or purifiedby affinity purification using an antibody of the invention. Methods ofenriching a sample for a protein or purifying a protein using affinitypurification are known in the art. In short, affinity purificationcomprises incubating a sample with a solid support, such as beads, aculture plate, or a membrane, that facilitates later steps. A solidsupport may be coated with an antibody of the invention, causing anacetaminophen-protein adduct to attach to the solid support.Alternatively, a sample may be incubated with an antibody of theinvention, and the acetaminophen-protein adduct-antibody complex may beisolated by incubating with a solid support coated with a secondantibody with specificity to an antibody of the invention, causing aprotein-antibody complex to attach to the solid support. Anacetaminophen-protein adduct may then be purified or enriched by washingother material in the sample that is not bound to the solid support, or,if the solid support is superparamagnetic beads, anacetaminophen-protein adduct attached to the beads (expressing theantigen) may be separated from the sample by attraction to a strongmagnetic field. Upon enrichment or purification, anacetaminophen-protein adduct may then be detected in the enriched orpurified sample using any of the methods described above.

In another embodiment, protein-specific antibodies may be used tocapture and isolate adducted protein(s), and then anacetaminophen-protein adduct antibody of the disclosure may be used todetect the adduction of the protein. Suitable protein-specificantibodies may be antibodies that specifically bind a protein known tobe modified with NAPQI. Non-limiting examples of proteins modified byNAPQI include betaine-homocysteine S-methyltransferase 1 (BHMT),cytoplasmic aspartate aminotransferase (cAspAT),1,4-alpha-glucan-branching enzyme, formimidoyltransferase-cyclodeaminase(FTCD), dystrophin, aldehyde dehydrogenase, ATP synthase alpha-chainmitochondrial, calregulin, carbamoylphosphate synthetase I, carbonatedehydratase III (CA-III), aldehyde dehydrogenase (AHD-M1), glutamatedehydrogenase (GDH), glutamate-ammonia ligase, cellular glutathioneperoxidase, glutathione transferases (GST), glutathione S-transferase P1, GAPDH, AdoMet synthetase 1, macrophage 23 kDa stress protein,eIF-4A-I, 56 kDa acetaminophen-binding protein, L-iditol2-dehydrogenase, amine N-methyltransferase, antioxidant protein 1,tropomyosin 3, urate oxidase, 10-formyltetrahydrofolate dehydrogenase,hemoglobin, 56 kDa selenium-binding protein, lamin A, cellular thyroidhormone binding protein, 58 kDa microsomal protein, Life Tech mouseembryo 8 5dpc 10664019 Mus musculus cDNA clone, inorganicpyrophosphatase, NML Mus musculus cDNA clone, 2-4-dienoyl-CoA reductasemitochondrial, 3-HAI,3-hydroxyanthranilate 3-4-dioxygenase, 94 kDaglucose-regulated protein, cytosolic inhibitor of Nrf2, serum albumin,and delayed early response protein 6. One or more adducted proteins maybe isolated and then an acetaminophen-protein adduct antibody of thedisclosure may be used to detect the amount of adducted protein asdescribed above.

(b) Methods to Detect Acetaminophen-Induced Toxicity in a Subject

In aspect, the disclosure provides means to classify a subject based onthe amount of acetaminophen-protein adduct measured in a biologicalsample obtained from the subject. The method generally comprises (i)measuring the amount of acetaminophen-protein adduct in a biologicalsample obtained from the subject using an antibody that specificallybinds acetaminophen-protein adduct, (ii) comparing the amount ofacetaminophen-protein adduct in the sample to a reference value, and(iii) classifying the subject as having a high or low amount ofacetaminophen-protein adduct based on the amount ofacetaminophen-protein adduct measured in the sample. Optionally, themethod may comprise (i) obtaining a biological sample from a subject andmeasuring the amount of acetaminophen-protein adduct in the sample usingan antibody that specifically binds acetaminophen-protein adduct, (ii)comparing the amount of acetaminophen-protein adduct in the sample to areference value, and (iii) classifying the subject as having a high orlow amount of acetaminophen-protein adduct based on the amount ofacetaminophen-protein adduct measured in the sample. In the foregoingmethodologies, one or more acetaminophen protein adducts may bemeasured. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 acetaminophenprotein adducts may be measured. Methods for obtaining a biologicalsample from a subject and measuring the amount of acetaminophen-proteinadduct in the sample using an antibody that specifically bindsacetaminophen-protein adduct are detailed above. In a preferredembodiment, the biological sample is biological fluid selected from thegroup consisting of blood, plasma, serum, urine and saliva. In aspecific embodiment, the acetaminophen-protein adduct is3-(cystein-S-yl)acetaminophen-protein adduct.

Any suitable reference value known in the art may be used. For example,a suitable reference value may be the amount of acetaminophen-proteinadduct in a biological fluid sample obtained from a subject or group ofsubjects of the same species that has normal hepatic function. Inanother example, a suitable reference value may be the amount ofacetaminophen-protein adduct in a biological fluid sample obtained froma subject, or group of subjects, of the same species that has nodetectable acetaminophen-induced toxicity. In another example, asuitable reference value may be the amount of acetaminophen-proteinadduct in biological fluid sample obtained from a subject or group ofsubjects of the same species that has acetaminophen-induced toxicity asmeasured by AST, ALT, bilirubin, INR or other non-specific biomarkers ofhepatic function. For example, a suitable reference value may be theamount of acetaminophen-protein adduct in a biological sample obtainedfrom a subject or group of subjects of the same species that hasacetaminophen-induced toxicity as measured by ALT levels >1000 IU. Inanother example, a suitable reference value may be the background signalof the assay as determined by methods known in the art. In anotherexample, a suitable reference value may be a measurement of the amountof acetaminophen-protein adduct in a reference sample obtained from thesame subject. The reference sample comprises the same type of biologicalfluid as the test sample, and may or may not be obtained from thesubject when hepatic function was normal. A skilled artisan willappreciate that it is not always possible or desirable to obtain areference sample from a subject when the subject is otherwise healthy.For example, in an acute setting, a reference sample may be the firstsample obtained from the subject at presentation. In another example,when monitoring the effectiveness of a therapy, a reference sample maybe a sample obtained from a subject before therapy began. In such anexample, a subject may have suspected acetaminophen-induced toxicity butmay not have other symptoms of acetaminophen-induced toxicity or thesubject may have suspected acetaminophen-induced toxicity and one ormore other symptom of acetaminophen-induced toxicity. In a specificembodiment, a suitable reference value may be a threshold previouslydetermined via other methods. For example, a suitable reference valuemay be a value corresponding to 1 nmol/ml of acetaminophen-proteinadduct as measured by high pressure liquid chromatography withelectrochemical detection (HPLC-EC).

According to the disclosure, a subject may be classified based on theamount of acetaminophen-protein adduct measured in the sample.Classifying a subject based on the amount of acetaminophen-proteinadduct measured in a sample of biological fluid obtained from thesubject may be used to identify subjects with acetaminophen-inducedexposure and/or toxicity. The term “acetaminophen-induced toxicity” isdescribed in detail below. Generally speaking, a subject may beclassified as having a high or low amount of acetaminophen-proteinadduct compared to a reference value, wherein a high amount ofacetaminophen-protein adduct is an amount above the reference value anda low amount is an amount equal to or below the reference value. Inpreferred embodiments, to classify a subject as having a high amount ofacetaminophen-protein adduct, the amount of acetaminophen-protein adductin the sample compared to the reference value may be at least 5%greater. For example, the amount of acetaminophen-protein adduct in thesample may be at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 100% greater than the reference value. In otherembodiments, the amount of acetaminophen-protein adduct in the sample ofbiological fluid obtained from the subject compared to the referencevalue may be increased at least 2-fold. For example, the amount ofacetaminophen-protein adduct in the sample compared to the referencevalue may be increased at least 2-fold, at least 5-fold, at least10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or atleast 50-fold.

In another aspect, the disclosure provides means to detectacetaminophen-induced exposure and/or toxicity in a subject. As usedherein, the term “acetaminophen-induced toxicity” refers to damage ordestruction to the liver due to acetaminophen. Acetaminophen, when takenin overdoses and sometimes even when introduced within therapeuticranges, may injure the liver. Damage to the liver is not due to the drugitself but to a toxic metabolite (N-acetyl-p-benzoquinone imine NAPQI,or NABQI) produced by cytochrome P-450 enzymes in the liver. In anoverdose, a large amount of NAPQI is generated, which overwhelms thedetoxification process and leads to liver cell damage. The risk of liverinjury is influenced by several factors including the dose ingested,concurrent alcohol or other drug intake, interval between ingestion andantidote, etc. The dose toxic to the liver is quite variable from personto person and is smaller in chronic alcoholics.

The causes of hepatotoxity known in the art are numerous, and mayinclude, but are not limited to, trauma, neoplastic disease, bacterialor viral infection, exposure to toxins, poisons, environmental, or othersubstances. Biomarkers of liver function are well known in the art.Non-limiting examples of biomarkers of liver injury include elevatedAST, ALT, bilirubin and PT (INR). However, increasedacetaminophen-protein adduct in a biological fluid may prove thatacetaminophen caused or contributed to the liver injury.

In addition to the detection of acetaminophen-induced toxicity, itshould also be appreciated by those of skill in the art that a method ofthe disclosure may be used to diagnose various features of treatmentwith acetaminophen and acetaminophen toxicity. A method of thedisclosure may be used to determine levels of acetaminophen intake by asubject to determine compliance with treatment. Alternatively, a methodof the disclosure may be used to determine the severity of acetaminophentoxicity. For instance, a method of the disclosure may be used todetermine normal sub-toxic levels of acetaminophen, thereby ruling outacetaminophen toxicity. A method of the disclosure may also be used todiagnose acetaminophen toxicity with good prognosis that will resolve.Alternatively, a method of the disclosure may be used to diagnoseacetaminophen toxicity with bad prognosis that will lead to death or theneed for a liver transplant. A method of the disclosure may also be usedto determine chronic acetaminophen exposure. As used herein, the term“chronic acetaminophen exposure” may be used to describe acetaminophentoxicity caused by exposure to repeated supratherapeutic acetaminophenover extended periods of time, such as, for instance, through ingestingsupratherapeutic doses of acetaminophen, or use of sustained releaseacetaminophen formulations. Additionally, a method of the disclosure maybe used to determine acute acetaminophen exposure. As used herein, theterm “acute acetaminophen exposure” may be used to describeacetaminophen toxicity caused by ingestion of a single large dose ofacetaminophen.

A method of the present disclosure may be used in combination with othermethods of diagnosing acetaminophen toxicity, or other clinicaldiagnostic methods. Additionally, a method of the present disclosure mayfurther comprise treatment of a subject. Non-limiting examples ofstandard treatments for acetaminophen toxicity administration ofactivated charcoal, administration of N-acetylcysteine (oral or IV),liver transplantation, and combinations thereof.

For each aspect, the method generally comprises (i) measuring the amountof acetaminophen-protein adduct in a biological sample obtained from asubject using an antibody that specifically binds acetaminophen-proteinadduct, and (ii) comparing the amount of acetaminophen-protein adduct inthe sample to a reference value. Optionally, the method may comprise (i)obtaining a biological sample from a subject, (ii) measuring the amountof acetaminophen-protein adduct in the sample using an antibody thatspecifically binds acetaminophen-protein adduct, and (iii) comparing theamount of acetaminophen-protein adduct in the sample to a referencevalue. A greater amount of acetaminophen-protein adduct in the samplecompared to the reference value indicates acetaminophen-inducedtoxicity. The amount of acetaminophen-protein adduct may be aqualitative, a semi-quantitative or quantitative measurement. Suitableanti-acetaminophen-protein adduct antibodies are described above, as aremethods for measuring the amount of acetaminophen-protein adduct in abiological sample. In a preferred embodiment, the biological sample isbiological fluid selected from the group consisting of blood, plasma,serum, urine and saliva.

III. Acetaminophen-Protein Adduct Immunogen

Another aspect of the present disclosure provides anacetaminophen-protein adduct immunogen for the production of antibodieswith specificity for acetaminophen-protein adducts. The novel immunogenwas prepared by modifying an immunogenic carrier protein (CP) with2-iminothiolane (2-IT) to provide a highly substituted CP with numerous5-carbon linker molecules with terminal sulfhydryl groups. This 2-ITmodified CP was then covalently modified at the terminal sulfhydrylgroups by reaction with biosynthetically preparedN-acetyl-p-benzoquinone imine (NAPQI). In a specific embodiment, theimmunogen is Carrier Protein-2-iminothiolane linked-actaminophenimmunogen. Accordingly, the immunogen may be referred to asCP-2-IT-APAP.

As used herein, a “carrier protein” is any protein used for couplingwith peptides or other haptens that are not sufficiently large orcomplex on their own to induce an immune response and produceantibodies. The carrier protein, because it is large and complex,confers immunogenicity to the conjugated hapten, resulting in antibodiesbeing produced against epitopes on the hapten and carrier. Many proteinscan be used as carriers and are chosen based on immunogenicity,solubility, and availability of useful functional groups through whichconjugation with the hapten can be achieved. Non-limiting examples ofsuitable carrier proteins include keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA), Blue Carrier Protein (Concholepasconcholepas hemocyanin (CCH)) and ovalbumin (OVA).

2-iminothiolane may also be referred to as 2-IT or Traut's reagent.2-iminothiolane is a small thiolation compound that reacts with primaryamines to add a small spacer arm (8.1 angstroms) terminated by a freesulfhydryl group. 2-iminothiolane is a cyclic thioimidate compound forthiolation (sulfhydryl addition). 2-IT reacts with primary amines (—NH₂)to introduce sulfhydryl (—SH) groups while maintaining charge propertiessimilar to the original amino group. Other linkers in place of 2-IT maybe used in an immunogen of the invention provided the linker contains asulfur bound at the carbon 3 position of the ring structure ofacetaminophen. The presence of the sulfur is essential to forming anantibody of the invention. The linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 carbons. In a specific embodiment, the linker may be 3, 4, 5 or 6carbons. In an exemplary embodiment, the linker is 5 carbons.

The carrier protein conjugated to a linker is reacted with NAPQI bymethods common in the art. In a specific embodiment, the CP-2-IT isreacted with NAPQI by methods common in the art. It is essential thatthe sulfhydryl group of the linker attached to CP be reacted with NAPQIto obtain an immunogen of the invention. In a specific embodiment, thesulfhydryl group of the 2-IT may be targeted for reaction with NAPQI.

The inventors have discovered that immunization with CP-2-IT-APAP mayproduce monoclonal antibodies with specificity for acetaminophen-proteinadducts. Specifically, immunization with CP-2-IT-APAP may producemonoclonal antibodies with specificity for 3-(cystein-S-yl)acetaminophenprotein adduct. Methods of making a monoclonal antibody using animmunogen of the invention are described in Section I. Using animmunogen of the invention, a monoclonal antibody may bind to anacetaminophen protein adduct approximately 2000 to 3000 times moreeffectively than free acetaminophen. In another embodiment, a monoclonalantibody may bind to an acetaminophen protein adduct approximately 8000times more effectively than free acetaminophen. For example, amonoclonal antibody may bind to an acetaminophen protein about 100,about 250, about 500, about 1000, about 1500, about 2000, about 2500,about 3000, about 3500, about 4000, about 4500, about 5000, about 5500,about 6000, about 6500, about 7000, about 7500, about 8000, about 8500,about 9000, about 9500, about 10,000, about 11,000, about 12,000, about13,000, about 14,000, about 15,000, about 16,000, about 17,000, about18,000, about 19,000 about 20,000, about 30,000, about 40,000, or about50,000 times more effectively than free acetaminophen.

Definitions

As used herein, “antibody” refers to an immunoglobulin derived moleculethat specifically recognizes acetaminophen-protein adduct. An antibodyof the invention may be a full length antibody (IgM, IgG, IgA, IgE) ormay be an antibody fragment (Fab, F(ab′)2, scFv). An antibody may bechimeric or may be humanized.

As used herein, “CDR” means “complementary determining region.” CDRs mayalso be referred to as hypervariable regions.

As used herein, “light chain” is the small polypeptide subunit of theantibody. A typical antibody comprises two light chains and two heavychains.

As used herein, the “heavy chain” is the large polypeptide subunit ofthe antibody. The heavy chain of an antibody contains a series ofimmunoglobulin domains, with at least one variable domain and at leastone constant domain.

“Humanized”, as used herein, refers to the process where monoclonalantibodies are produced using recombinant DNA to create constructscapable of expression in human cell culture. Any known techniques forproducing these constructs will work for purposes of the presentinvention.

As used herein, “single chain variable fragments” or “scFv” or “scFvs”,refer to fusion proteins of the variable regions of the heavy and lightchains of immunoglobulins connected via a linker. In some embodiment,the linker is a peptide of about 10 to 25 amino acids.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1. Production of Monoclonal Antibodies Specific forAcetaminophen-Protein Adduct

The hepatotoxicity of acetaminophen (APAP) (also called paracetamol) ismediated by the reactive metabolite N-acetyl-p-benzoquinone imine whichbinds covalently to protein as 3-(cystein-S-yl)acetaminophen. Theseacetaminophen-protein adducts are specific biomarkers of exposure toacetaminophen and elevated levels of these adducts are a specificbiomarker of acetaminophen toxicity. This disclosure is to describe anew and unique immunogen for the preparation of monoclonal antibodieswith specificity for the acetaminophen-protein adduct. The resultantantibodies react specifically with the acetaminophen-protein adductsthat are formed physiologically during the pathogenesis ofacetaminophen-mediated toxicity.

The inventors conceived of and synthesized a new immunogen for thepurpose of preparing antibodies with specificity for acetaminophenprotein adducts. The new immunogen was prepared by modifying animmunogenic carrier protein (CP) with 2-iminothiolane (2-IT) to providea highly substituted CP with numerous 5-carbon linker molecules withterminal sulfhydryl groups. This 2-IT modified CP was then covalentlymodified at the terminal sulfhydryl groups by reaction withsynthetically prepared N-acetyl-p-benzoquinone imine. For shorthandreference, the Carrier Protein-2-iminothiolane linked-acetaminophenimmunogen is referred to as CP-2-IT-APAP. Immunizing rabbits withCP-2-IT-APAP resulted in the production of polyclonal rabbit antibodieswith specificity for the physiologically formed3-(cystein-S-yl)acetaminophen protein adducts and this was confirmed byELISA and Lateral Flow Immunoassay using acetaminophen proteins adductsas solid-phase antigen. Subsequently, the CP-2-IT-APAP immunogen wasused to prepare rabbit monoclonal antibodies (mAb) with specificity foracetaminophen-protein adducts. To confirm binding of purified antibodiesto acetaminophen-protein adducts, an ELISA was performed. In the ELISAexperiment, antigen is coated overnight at 4° C. Samples are added inserial dilutions starting at 1:250 (supernatant and flow-through) or 4μg/ml (purified antibody) and incubated at room temperature for 1.5hours. Goat anti-rabbit alkaline phosphatase-conjugated secondaryantibody is added at room temperature for 1 hour. Substrate solution isadded and developed for 15 minutes at room temperature. Absorbance ismeasured at 405 nm. Data in FIG. 1 represents the average of repetitions(rep) 1 and 2 for each sample.

Example 2. Competitive ELISA to Determine the Relative InhibitoryPotency of APAP Bound to Protein as APAP-Protein Adduct Versus FreeUnbound APAP

Tissue culture supernatants from Rabbit monoclonal antibody clone 14-12at a dilution of 1:250 was mixed with serial 4-fold dilutions ofinhibitor (either BSA-APAP or APAP) such that the final dilution of RmAbwas 1:500. The final concentrations of APAP were 80, 20, 5, 1.25, and0.31 nmole per ELISA well. The final concentrations of APAP-BSA(quantified by HPLC-EC as APAP-Cys from the hydrolyzed protein) were 10,2.5, 0.625, 0.156, and 0.04 pmole per ELISA well.

Selection of promising RmAb clones for future use to detectacetaminophen protein adducts was based on efficiency of immunoglobulinproduction, affinity for detection of APAP-protein adduct(3-(cystein-S-yl)acetaminophen), and relative insensitivity fordetection of the free drug APAP.

Synthetically prepared acetaminophen-protein adduct (BSA-APAP) and freedrug (APAP) were evaluated in competitive ELISA to determine theirrelative capacity, on a molar basis, to inhibit the binding of clone14-12 rabbit monoclonal antibody to solid-phase immobilizedacetaminophen protein adduct. Data is plotted as percent inhibition andindicate that it takes approximately 8,000 free APAP molecules toproduce the same inhibitory potency as one molecule of APAP-Cys asprotein adduct (FIG. 2). Restated, clone 14-12 antibody hasapproximately 8000 times more affinity for acetaminophen protein adductthan it does for free APAP as measured in this ELISA context.

The competitive ELISA was repeated with rabbit monoclonal antibody(RMAb) 14-12 and rabbit monoclonal antibody (RMAb) 22-8. Briefly, ELISAplates were coated with BSA-APAP, 200 ng (protein)/well. RMAb clonesubernatant (1:250 dilution) was combined with an equal volume of serial4-fold dilutions of inhibitor to give a final antibody dilution of 1:500and the indicated final concentrations of APAP-BSA and APAP. BSA-APAPprepared by reacting bovine serum albumin (BSA) withN-Acetyl-p-benzoquinone imine (NAPQI) to form 3-(cystein-S-yl)acetaminophen protein adducts on BSA (BSA-APAP). After incubation andwashing, bound RMAb was detected using Goat anti-Rabbit-IgG conjugatedto HRP followed by the substrate TMB and color development wasdetermined using an ELISA plate reader. Dilution Buffer was 0.025% (w/v)non-fat milk protein in phosphate buffered saline containing 0.15 MNaCl, pH 7.4.

As demonstrated above, clone 14-12 antibody has approximately 8000 timesmore affinity for acetaminophen protein adduct than it does for freeAPAP as measured in this ELISA context. Additionally, clone 22-8antibody has approximately 1250 times more affinity for acetaminophenprotein adduct than it does for free APAP as measured in this ELISAcontext (FIG. 3).

Example 3. Binding of RMAb to Acetaminophen Protein Adduct Immobilizedat Test Band in Lateral Flow Assays

Binding of RMAb to acetaminophen protein adduct immobilized at the testband of lateral flow assays was determined by preparing serial dilutionsof RMAb in dilution buffer (phosphate buffered saline containing, 0.02%NaN₃ and 0.125% (WN) non-fat dry milk). Bound RMAb was detected using 40nm nanoparticulate gold adsorbed on Goat anti-Rabbit IgG. The log plotof RMAb (μg/ml IgG) versus Test Band Reading (arbitrary reflectanceunits) indicates that 0.01 μg RMAb gives a Test Band reflectance ofapproximately 20,000 (FIG. 4). A value of 0.01 μg was calculated basedon that 100 μl of a 0.1 μg/ml solution was used. Subsequent competitiveinhibition assays in lateral flow format used this amount of RMAb. TestBand antigen was ovalbumin modified with NAPQI to produce APAP-proteinadduct.

Example 4. Competitive Lateral Flow Immunoassay with RMAb Clones 14-12and 22-8: Relative Potency of Parent Drug (APAP) Versus Adduct(APAP-Protein) as Inhibitor

A competitive inhibition assay using RMAb in lateral flow format wasthen performed. RMAb was diluted to 0.2 μg/ml and this Ab concentrationwas combined with an equal volume of inhibitor, either BSA-APAP or APAP,such that the final concentration applied to each 100 μl lateral flowassay was 0.01 μg RMAb and the indicated final concentration ofinhibitor. The data indicate that the lateral flow assay using RMAbdetected APAP-protein adduct in the range of seven serial 2-folddilutions from 1.19 to 0.0186 μM and detected APAP in the range sixserial 2-fold dilutions of 2500 to 78 μM (final concentrations) (FIG.5). Collectively the data indicates that the assay is sensitive for thedetection of APAP-protein adducts (APAP-Cys) and much less sensitive(>8000-fold) for the detection of APAP. Restated: one mole of APAP-Cysis approximately 8000 times more potent than one mole of APAP for theinhibition of RMAb binding to the APAP-protein adduct immobilized at thetest band.

Next, the same competitive assay was performed but instead theAPAP-protein adduct was physiologically formed APAP-protein adduct fromthe serum of an APAP toxicity patient. APAP protein adduct concentrationof the inhibitor was determined by HPLC-EC. Human APAP-protein adductand APAP were diluted in control human serum. The data againdemonstrated that the assay is sensitive for the detection ofAPAP-protein adducts (APAP-Cys) and much less sensitive for thedetection of APAP (FIG. 6).

Example 5. Development of Immunoassays for Acetaminophen Toxicity

The identification of NAPQI adducted proteins may allow development ofspecific immunoassays for acetaminophen toxicity. In one embodiment,protein-specific antibodies may be used in a competitive immunoassay inwhich a limiting amount of antibody specific for acetaminophen-proteinadduct may be mixed with a sample putatively containingacetaminophen-protein adducts and, if present in the sample, the adductswill inhibit the binding of antibody to an immobilized syntheticallyprepared acetaminophen-protein adduct. This method would measure totaladducts (including all acetaminophen-protein adducts regardless of what(cysteine-containing) proteins in the sample were adducted. Typicalexamples would include the ELISA in FIG. 2 and the Lateral Flow assay inFIG. 5 and FIG. 6.

In another embodiment, protein-specific antibodies may be used tocapture and isolate adducted protein(s), and then a second antibodyspecific for acetaminophen-cysteine adducts (total adducts) may be usedto detect the adduction of the protein.

Human acetaminophen overdose and exposure samples may be analyzed tounderstand the frequency of occurrence of the specific protein adductsamong different degrees of severity or circumstances of toxicity. Toaccomplish this, additional methodology may be developed toantibody/affinity isolate specific proteins that contain cysteine andare thus candidates for adduct formation and thus enrich for thespecific adduct proteins from human samples. For example, assays usingsolid-phase antibodies to a specific protein (on paramagnetic beads orother solid phase matrix) to capture the specific protein may beperformed and complimented with detection of adduct proteins using themonoclonal antibodies produced in Example 1 with specificity for APAPbound to protein. Essentially the assay may involve interrogating theadducted protein two times: 1) capture by specific anti-proteinantibody, and 2) detection with the antibodies specific for the haptenprotein linkage produced in Example 1. Commercially availableanti-protein antibodies, or newly-developed antibodies designedspecifically for the use described herein, may be used.

Preferably, the assay may involve interrogating the adducted protein bycapturing with monoclonal antibodies produced in Example 1 withspecificity for the hapten protein linkage, and detecting withantibodies specific for the protein.

What is claimed is:
 1. An isolated antibody, wherein the antibodyspecifically binds an acetaminophen-protein adduct but does notspecifically bind free acetaminophen and recognizes the immunogen:Carrier Protein-2-iminothiolane-APAP, wherein the antibody comprises:(a) a light chain CDR1 amino acid sequence of SEQ ID NO:21; (b) a lightchain CDR2 amino acid sequence of SEQ ID NO:22; (c) a light chain CDR3amino acid sequence of LGh; wherein h is a hydrophobic amino acid; (d) aheavy chain CDR1 amino acid sequence of YXI, wherein X is any aminoacid; (e) a heavy chain CDR2 amino acid sequence of SEQ ID NO:23; and(f) a heavy chain CDR3 amino acid sequence of SEQ ID NO:24.
 2. Theisolated antibody of claim 1, wherein the antibody comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:14 and SEQID NO:16.
 3. The isolated antibody of claim 1, wherein the antibodycomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:13 and SEQ ID NO:15.
 4. The isolated antibody of claim 1,wherein the antibody is encoded by a nucleic acid sequence comprising anucleic acid sequence selected from the group consisting of SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.
 5. An isolatedantibody, wherein the antibody specifically binds anacetaminophen-protein adduct but does not specifically bind freeacetaminophen and comprises: (a) a light chain CDR1 amino acid sequenceof SEQ ID NO:21; (b) a light chain CDR2 amino acid sequence of SEQ IDNO:22; (c) a light chain CDR3 amino acid sequence of LGh; wherein h is ahydrophobic amino acid; (d) a heavy chain CDR1 amino acid sequence ofYXI, wherein X is any amino acid; (e) a heavy chain CDR2 amino acidsequence of SEQ ID NO:23; and (f) a heavy chain CDR3 comprising theamino acid sequence of SEQ ID NO:6 with zero to two amino acidsubstitutions or SEQ ID NO:12 with zero to two amino acid substitutions.6. An isolated antibody, wherein the antibody specifically bindsacetaminophen-protein adduct but does not specifically bind freeacetaminophen and comprises: (a) a light chain CDR1 amino acid sequenceof SEQ ID NO:21; (b) a light chain CDR2 amino acid sequence of SEQ IDNO:22; (c) a light chain CDR3 comprising the amino acid sequence of SEQID NO:3 with zero to two amino acid substitutions or SEQ ID NO:9 withzero to two amino acid substitutions; (d) a heavy chain CDR1 amino acidsequence of YXI, wherein X is any amino acid; (e) a heavy chain CDR2amino acid sequence of SEQ ID NO:23; and (f) a heavy chain CDR3 aminoacid sequence of SEQ ID NO:24.
 7. The isolated antibody of claim 1,wherein the antibody is selected from the group consisting of asingle-chain antibody, an antibody fragment, a chimeric antibody, or ahumanized antibody.
 8. The isolated antibody of claim 1, wherein theantibody specifically binds to an acetaminophen protein adduct more than2000 times more effectively than free acetaminophen.
 9. The isolatedantibody of claim 1, wherein the antibody specifically binds to anacetaminophen protein adduct about 8000 times more effectively than freeacetaminophen.
 10. The isolated antibody of claim 5, wherein theantibody is selected from the group consisting of a single-chainantibody, an antibody fragment, a chimeric antibody, or a humanizedantibody.
 11. The isolated antibody of claim 5, wherein the antibodyspecifically binds to an acetaminophen protein adduct more than 2000times more effectively than free acetaminophen.
 12. The isolatedantibody of claim 5, wherein the antibody specifically binds to anacetaminophen protein adduct about 8000 times more effectively than freeacetaminophen.
 13. The isolated antibody of claim 6, wherein theantibody is selected from the group consisting of a single-chainantibody, an antibody fragment, a chimeric antibody, or a humanizedantibody.
 14. The isolated antibody of claim 6, wherein the antibodyspecifically binds to an acetaminophen protein adduct more than 2000times more effectively than free acetaminophen.
 15. The isolatedantibody of claim 6, wherein the antibody specifically binds to anacetaminophen protein adduct about 8000 times more effectively than freeacetaminophen.